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decrease in proliferation of only a subset of T cells, or an apparent decrease in total lymphocyte
proliferation due to T-cell lymphopenia and under-representation of T cells in the PBMC pool. None of
these can be discriminated by the thymidine uptake assay, but can be assessed by flow cytometry, which
uses antibodies to identify specific responder cell populations. Cell viability can also be measured
within the same assay without requiring additional cell manipulation or specimen. Mitogens are very
potent stimulators of T-cell activation and proliferation independent of their antigenic specificity.(3) It
has been suggested that mitogens can induce T-cell proliferative responses even if they are incapable of
responding adequately to antigenic (physiologic) stimuli. Therefore, abnormal T-cell responses to
mitogens are considered a diagnostically less sensitive but more specific test of aberrant T-cell function.
Lectin mitogens have been shown to bind the T-cell receptor, which is glycosylated through its
carbohydrate moiety, thereby activating quiescent T cells. Mitogenic stimulation has been shown to
increase intracellular calcium (CA++) in T cells, which is absolutely essential for T-cell proliferation.
While PHA is a strong T-cell mitogen, PWM is a weak T-cell mitogen, but it also induces B-cell
activation and proliferation as well. For this assay, we use a method that directly measures the S-phase
proliferation of lymphocytes through the use of Click chemistry. In the Invitrogen Click-iT-EdU assay,
the Click chemistry has been adapted to measure cell proliferation through direct detection of nucleotide
incorporation. In the assay, an alkyne-modified nucleoside is supplied in cell-growth media for a
defined time period and is incorporated within cells. The cells are subsequently fixed, permeabilized,
and reacted with a dye-labeled azide, catalyzed by copper. A covalent bond is formed between the dye
and the incorporated nucleotide, and the fluorescent signal is then measured by flow cytometry.(4)
Specific proliferating cell populations can be visualized by the addition of cell-specific antibodies. Cell
viability, apoptosis, and death can also be measured by flow cytometry using 7-AAD and Annexin V.
The Click-iT-EdU assay has already been shown to be an acceptable alternative to the 3H-thymidine
assay for measuring lymphocyte/T-cell proliferation.(5) The absolute counts of lymphocyte subsets are
known to be influenced by a variety of biological factors, including hormones, the environment, and
temperature. The studies on diurnal (circadian) variation in lymphocyte counts have demonstrated
progressive increase in CD4 T-cell count throughout the day, while CD8 T cells and CD19+ B cells
increase between 8:30 am and noon, with no change between noon and afternoon. Natural killer
(NK)-cell counts, on the other hand, are constant throughout the day. Circadian variations in circulating
T-cell counts have been shown to be negatively correlated with plasma cortisol concentration. In fact,
cortisol and catecholamine concentrations control distribution and, therefore, numbers of naive versus
effector CD4 and CD8 T cells. It is generally accepted that lower CD4 T-cell counts are seen in the
morning compared with the evening, and during summer compared to winter. These data, therefore,
indicate that timing, and consistency in timing, of blood collection is critical when serially monitoring
patients for lymphocyte subsets.
Useful For: Assessing T-cell function in patients on immunosuppressive therapy, including solid-organ
transplant patients Evaluating patients suspected of having impairment in cellular immunity Evaluation of
T-cell function in patients with primary immunodeficiencies, either cellular (DiGeorge syndrome,
T-negative severe combined immunodeficiency [SCID], etc) or combined T- and B-cell
immunodeficiencies (T- and B-negative SCID, Wiskott Aldrich syndrome, ataxia telangiectasia, common
variable immunodeficiency, among others) where T-cell function may be impaired Evaluation of T-cell
function in patients with secondary immunodeficiency, either disease related or iatrogenic Evaluation of
recovery of T-cell function and competence following bone marrow transplantation or hematopoietic stem
cell transplantation
Interpretation: Abnormal test results to mitogen stimulation are indicative of impaired T-cell function
if T-cell counts are normal or only modestly decreased. If there is profound T-cell lymphopenia, it must
be kept in mind that there could be a "dilution" effect with under-representation of T cells within the
peripheral blood mononuclear cells (PBMCs) population that could result in lower T-cell proliferative
responses. However, this is not a significant concern in the flow cytometry assay, since acquisition of
additional cellular events during analysis can compensate for artificial reduction in proliferation due to
lower T-cell counts. There is no absolute correlation between T-cell proliferation in vitro and a clinically
significant immunodeficiency, whether primary or secondary, since T-cell proliferation in response to
activation is necessary, but not sufficient, for an effective immune response. Therefore, the proliferative
response to mitogens can be regarded as a more specific but less sensitive test for the diagnosis of
infection susceptibility. It should also be kept in mind that there is no single laboratory test that can
identify or define impaired cellular immunity, with the exception of an opportunistic infection. Controls in
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